Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars

The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2–6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2–G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations—sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.     

Purification and properties of cellobiose phosphorylase from Clostridium thermocellum

Cellobiose phosphorylase was purified 111-fold from a cell extract of Clostridium thermocellum ATCC 27405, with a yield of 31.4%, to electrophoretic and column chromatographic homogenity. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consisted of two identical subunits. It was suggested by spectrophotometric and chemical analyse that the enzyme contained no pyridoxal phosphate. The enzyme was inactivated by N-ethylmaleimide and activated by dithiothreitol, indicating that the exposed thiol group(s) was important for the enzymatic activity. The enzyme could utilize, so far as examined, d-glucose, d-xylose, 2-deoxy-d-glucose, and d-mannose, as acceptors of glucose in the synthetic reaction of disaccharides. The enzyme could to a low degree utilize d-arabinose and d-fucose, as acceptors.     

Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth. The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C. The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively). Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C). Growth on cellobiose was associated with induction of a cellobiose permease activity. In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates. The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively. The molar yields were 38 on cellobiose and 20 on glucose. Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities. The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.     

Utilization of cellobiose and D-glucose by Clostridium thermocellum ATCC-27405

Cultures of Clostridium thermocellum ATCC-27405, maintained on cellulose and not adapted to grow on glucose utilize cellobiose preferentially over D-glucose, and are only able to initiate growth on D-glucose when the cellobiose has been exhausted from the growth medium. However, D-glucose is the carbon source preferentially utilized when cultures of this microorganism, previously adapted for growth on glucose, are transferred to a medium with equivalent concentrations of both sugars. One reason for the preferential utilization of glucose over that of cellobiose might be the competitive inhibition of cellobiose phosphorylase by intracellular glucose accumulation. When in the glucose-adapted cultures the pressure to grow on glucose as the sole carbon source is again released, both sugars can be simultaneously utilized.     

Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola.

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

Engineering a large protein by combined rational and random approaches: stabilizing the Clostridium thermocellum cellobiose phosphorylase

The Clostridium thermocellum cellobiose phosphorylase (CtCBP) is a large protein consisting of 812 amino acids and has great potential in the production of sugar phosphates, novel glycosides, and biofuels. It is relatively stable at 50 °C, but is rapidly inactivated at 70 °C. To stabilize CtCBP at elevated temperatures, two protein-engineering approaches were applied, i.e. site-directed mutagenesis based on structure-guided homology analysis and random mutagenesis at various mutation rates. The former chose substitutions by comparison of the protein sequences of CBP homologs, utilized structural information to identify key amino acid residues responsible for enhanced stability, and then created a few variants accurately. The latter constructed large libraries of random mutants at different mutagenesis frequencies. A novel combinational selection/screening strategy was employed to quickly isolate thermostability-enhanced and active variants. Several stability-enhanced mutants were obtained by both methods. Manually combining the stabilizing mutations identified from both rational and random approaches led to the best mutant (CM3) with the halftime of inactivation at 70 °C extended from 8.3 to 24.6 min. The temperature optimum of CM3 was increased from 60 to 80 °C. These results suggested that a combination of rational design and random mutagenesis could have a solid basis for engineering large proteins.     

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum

 Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam^- phenotype. Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995     

Kinetics of the extracellular cellulases of Clostridium thermocellum acting on pretreated mixed hardwood and Avicel

Initial hydrolysis rates were examined for mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220 °C (PTW220) and Avicel. Linear rates were observed for fractional conversion relative to the theoretical up to 0.2 for PTW220 and 0.4 for Avicel. Initial rates were essentially unaffected by the presence of growth medium components over a range of pH values. Avicel-hydrolyzing activity was inhibited linearly by ethanol, with a 50% rate reduction at 8 wt.% ethanol. Rate saturation with either substrate or enzyme was observed in a manner qualitatively consistent with previously reported adsorption data. Although somewhat less reactive than Avicel at very low enzyme loadings, much higher reaction rates were observed for PTW220 at moderate and high enzyme loading because of its higher capacity to bind cellulase. At equal subtrate concentrations (as potential glucose) and fractional substrate coverage of 0.09, the initial rate of pretreated wood hydrolysis exceeded that of Avicel by 15-fold. For fractional substrate coverage values up to 0.09 (the maximum value achieved for PTW220), the initial rate was proportional to adsorbed enzyme for PTW220. However, the rate per adsorbed enzyme declined sharply with increasing fractional coverage for Avicel hydrolysis.     

Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli

A cellobiose phosphorylase (CBP) cloned from the Clostridium thermocellum YM4 strain was purified to homogeneity, and the reaction mechanisms of both the phosphorolytic and synthetic reactions were studied in detail. The enzyme reaction proceeded via an ordered bi bi mechanism, in which P(i) bound to the enzyme prior to D-cellobiose and then G 1-P was released after D-glucose. The order of substrate binding was different from that of CBP from Cellvibrio gilvus, which bound to cellobiose prior to P(i). In the synthetic reaction, the enzyme showed three times higher activity with beta-D-glucose than with alpha-D-glucose, and also showed weak activity with 1,5-anhydro-D-glucitol, indicating that the beta-anomeric hydroxyl group of D-glucose is highly required. However, even when it is removed enzyme activity remains. The substrate specificity and kinetic studies revealed that the configurations of the C3 and C4 hydroxyl groups were strictly required for the enzyme activity, whereas those of C2 and C6 could be substituted or deleted. The mechanism of substrate inhibition by D-glucose was studied in detail and it was concluded that D-glucose competed with G 1-P for its binding site in the synthetic reaction.     

Cellulolytic and physiological properties of Clostridium thermocellum

Three strains of Clostridium thermocellum obtained from various sources were found to have nearly identical deoxyribonucleic acid guanosine plus cytosine contents that ranged from 38.1–39.5 mole-%. All strain examined fermented only cellulose and cellulose derivatives, but not glucose, or xylose or other sugars. The principal cellulose fermentation products were ethanol, lactate, acetate, hydrogen and carbon dioxide. Growth of C. thermocellum on cellulose resulted in the production of extracellular cellulase that was non-oxygen labile, was thermally stable at 70° C for 45 min and adsorbed strongly on cellulose. Production of cellulase during fermentation correlated linearly with growth and cellulose degradation. Both the yield and specific activity of crude cellulase varied considerably with the specific growth substrates. Highest cellulase yield was obtained when grown on native cellulose, -cellulose and low degree of polymerization cellulose but not carboxymethylcellulose or other carbohydrate sources. Cellulase activity was not detected when cells were grown on cellobiose. Crude extracellular protein preparations lacked proteolytic and cellobiase activity. The pH and temperafure optima for endoglucanase activity were 5.2 and 65° C, respectively, while that of the exoglucanase activity were 5.4 and 64° C, respectively. The specific activity at 60° c for exoglucanase and endoglucanase of crude cellulase obtained from cells grown on cellulose (MN 300) was 3.6 moles reducing sugar equivalents released per h (unit)/mg of protein and 1.5 mole reducing sugar equivalent released per min (unit)/mg of protein, respectively. The yield of endoglucanase was 125 units per g of cellulose MN 300 degraded and that of exoglucanase was 300 units per g of cellulose MN 300 degraded. Glucose and cellobiose were the hydrolytic end products of crude cellulase action on cellulose, cellotraose and cellotriose in vitro.     

Form and Function of Clostridium thermocellum Biofilms

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h⁻¹) 12-fold higher than the bacterium''s maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.     

Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum

Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only β-1,3 and β-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G₂ to G₅), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G₃), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins (G₃ to G₅) are assimilated faster than cellobiose.     

Carbon balance and circadian regulation of hydrolytic and phosphorolytic breakdown of transitory starch

Transitory starch is formed in chloroplasts during the day and broken down at night. Transitory starch degradation could be regulated by light, circadian rhythms, or carbon balance. To test the role of these potential regulators, starch breakdown rates and metabolites were measured in bean (Phaseolus vulgaris) and Arabidopsis (Arabidopsis thaliana) plants. In continuous light, starch and maltose levels oscillated in a circadian manner. Under photorespiratory conditions, transitory starch breakdown occurred in the light faster than at night and glucose-6-P (G6P) was elevated. Nonaqueous fractionation showed that the increase in G6P occurred in the chloroplast. When Arabidopsis plants lacking the plastidic starch phosphorylase enzyme were placed under photorespiratory conditions, G6P levels remained constant, indicating that the increased chloroplastic G6P resulted from phosphorolytic starch degradation. Maltose was increased under photorespiratory conditions in both wild type and plants lacking starch phosphorylase, indicating that regulation of starch breakdown may occur at a point preceding the division of the hydrolytic and phosphorolytic pathways. When bean leaves were held in N2 to suppress photosynthesis and Suc synthesis without increasing photorespiration, starch breakdown did not occur and maltose and G6P levels remained constant. The redox status of the chloroplasts was found to be oxidized under conditions favoring starch degradation.     

Lipids and fatty acids in cellulosomes of Clostridium thermocellum

A lipid component was found in cellulosomes (multienzymatic cellulase complexes) of the thermophilic bacterium Clostridium thermocellum. Two major fractions of the cellulosomes have been studied, one with a relative molecular mass (M_r) of 10–50 million (polycellulosomes, fraction A) and the other with an M_r 0.5–10 million (fraction B) It was found that the larger cellulosomes contained higher relative amounts of lipids (8.1%) as well as Ca²⁺ ions (0.6%), and showed higher cellulolytic activity Among the lipids was cardiolipin, 1,2- and 1,3-diglycerides, triglycerides, and up to 11 free fatty acids, including both saturated (palmitic, lauric, myristic, pentadecanoic, stearic, arachinic) and unsaturated (myristoleic, palmitoleic, and oleic) moieies Cardiolipin was a major phospholipid component in cellulosomes and was also found to be a major phospholipid component of the cell membrane, palmitic acid was a major fatty acid Fraction B contained less fatty acids (0.5% vs 1.27% in fraction A) with fewer acids detected than in fraction A Removal of the extractable lipids led to fragmentation of the cellulosomes with a concurrent sharp drop in their enzymatic activity Total removal of the lipids from cellulosomes was possible only when the proteins were completely denatured The qualitative composition of the extractable and non-extractable fatty acids was the same The lipid component of the cellulosomes, containing a high content of the unsaturated fatty acids, was located mainly in the part of cellulosomes that is in tight contact with the cellulose surface, and it apparently plays an important role in the tight adsorption of the cellulosomes on cellulose.     

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Rates of phosphorolytic cleavage of β-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by ≥20-fold for both cellobiose and cellopentaose over a 10-fold range of β-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of β-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V_max values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K_m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).